rDNA sequentiation and phylogenetic analysis of the ITS region of strains of the brown-rot polypore Ryvardenia cretacea from Australia (Tasmania, Victoria) and Argentina (Patagonia) revealed the existence of two well supported clades. Each clade appears restricted to one side of the Pacific indicating a strong biogeographical isolation between strains and populations in the process of speciation. Nevertheless, detailed studies and comparisons did not show the existence of significant morphological differences. This fact, coupled with previously reported dikaryon x monosporic mating tests which showed biological compatibility between specimens from both regions, supports the maintenance of a complex but taxonomically single entity.

Ryvardenia Rajchenb. (Polyporales, Basidiomycota) is an austral genus of brown-rot polypores that was established in order to accommodate Polyporus cretaceus Lloyd (type species) and Polyporus campylus Berk. (Rajchenberg, 1994). The genus is characterized by basidiomes with an obscurely dimitic hyphal system in the dissepiments, where long,terminating, thick-walled hyphae resemble skeletal hyphae, while in the context the hyphal system appears to be monomitic; the tissues are characterized by the presence of thickwalled, clamped generative hyphae with glossy/refringent walls that are not metachromatic in cresyl-blue and that contrast with thinwalled generative hyphae; basidiospores are obovate, broad-ellipsoid to drop-shaped and thick-walled. From a biological point of view Ryvardenia is characterized by: brownrot taxa with a heterothallic, bipolar mating system; astatocoenocytic nuclear behavior of the mycelium (Boidin, 1971); and in culture generative hyphae of the advancing mycelium being simple-septate while towards the colony centre generative hyphae are homogeneously sclerified and clamped and fiber hyphae are never irregularly thick-walled (Rajchenberg, 1994).

Species in Ryvardenia were previously treated in different monomitic polypore genera such as Tyromyces P. Karst., Postia Fr., Spongipellis Pat., and Grifola S.F. Gray, or in the dimitic Piptoporus P. Karst. The genus was accepted by Buchanan & Ryvarden (2000) in their review of New Zealand polypores. Its validity from a phylogenetic perspective remains to be tested.

Ryvardenia cretacea (Lloyd) Rajchenb. is distinguished by its large, generally solitary basidiomes, with a consistency that turns chalky upon drying and, microscopically, by its generative hyphae of variable width that become tortuous, with swellings and with hyphal segments appearing as if disarticulated. It decays standing or fallen Nothofagus in southern Argentina and southern Chile and also Eucalyptus in SE Australia. It has been recorded as a wood-rotting species of minor importance for standing Nothofagus pumilio forests in southern Argentina (Cwielong & Rajchenberg, 1995). During a study of Postia s.l. taxa known from southern Argentina (Rajchenberg, 2006) we were able to compare several strains of R. cretacea from different geographic origins using molecular methods. The genus Ryvardenia has not until now been included in molecular phylogenetic analyses of brown-rot polypore genera and its phylogenetic relationships are, thus, unknown. The aim of the present work is to determine the variability of R. cretacea isolates based on the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA, on micro and macro-morphology and on sexuality; and to assess their phylogenetic relationships.

Material and Methods

Strains and dried specimens studied

Strains are deposited at and retrieved from the author's institutional culture collection (CIEFAPcc), from the International Collection of Micro-organisms from Plants (ICMP) at Landcare Research (Auckland, New Zealand) and from the Center for Forest Mycology Research Culture Collection and Herbarium (CFMR) at the Forest Products Laboratory (USDA Forest Service, Madison, WI, USA).

DNA from tissue cultures cited above, grown on malt extract liquid medium, was extracted using the UltraCleanTM Microbial DNA Isolation Kit (MO BIO laboratories inc., Solana Beach, USA) according to the manufacturer's instructions. The ITS region of the extracted DNA was amplified using the universal primers ITS5 and ITS4 (White et al., 1990).

PCR reaction mixtures for amplification of the ITS region were as described by Rajchenberg et al. (2011): dNTPs (0.25 mM of each), 2.5 mM MgCl2; PCR buffer supplied with the polymerase enzyme; 0.1 µM of each primer; 100-500 ng DNA; and 1.25 U of GoTaq polymerase (Promega, Madison, WI, USA). The final reaction volume was 50 µL. The PCR reactions were performed in a thermal cycler (My CyclerTM, BioRad) and the conditions were as described by Rajchenberg et al. (2011). PCR products were separated on a 1% (w/v) ethidium bromide-stained agarose gel and the bands were visualised under UV illumination. The amplified fragments were purified and sequenced at the DNA Synthesis and Sequencing Facility, Macrogen (Seoul, Korea). GenBank accession numbers of the sequences obtained are shown in Table 1.

Table 1 Fungal strains from Argentina and Australia used in this study, and GenBank sequences included in the phylogenetic analysis.

DNA sequences were aligned on MEGA version 4.0 (Tamura et al., 2007; available from the authors upon request). Phylogenetic trees based on neighbourjoining (NJ) and maximum parsimony (MP) were generated. A NJ tree was generated using MEGA version 4.0 (Tamura et al., 2007), with gaps and missing data deleted prior to the analysis. A maximum composite likelihood model was used to calculate the distances between taxa. Bootstrap analysis included 1,000 replicates using the same settings. MP trees were generated using PAUP* 4.0b10 (Swofford, 2000). For MP phylogenetic analyses, gaps were treated as missing characters and trees were generated by heuristic searches with random addition of sequences(1000 replicates), TBR (tree bisection reconnection) branch swapping and MULTREES effective. Bootstrapping (1000 bootstrap replicates) was used to determine confidence in the branches.

Herbarium specimens were studied with classical methods in order to corroborate morphological features described previously (Rajchenberg, 1994, 2006). This included the study of 30 spores of each collection, which were measured in sections mounted in Melzer's reagent; their mean length and width were determined in order to compare and/or distinguish differences in their sizes. One-way ANOVA analyses were performed using SPSS v.17

Results

The ITS-sequence dataset included 718 total characters, of which 98 were parsimony informative. Heuristic tree searches under MP analysis resulted in two equally most parsimonious trees. The tree length (TL) was 333, consistency index (CI) and retention index (RI) were 0.9 and 0.78, respectively. One of these trees is presented in Fig. 1. Bootstrap values obtained using MP and NJ were very similar and both analyses resulted in a similar tree topology, with high statistical support for the Ryvardenia cretacea species complex. Geographical sub-groups within R. cretacea were well supported; bootstrap values for Argentinean specimens (ARG): MP= 99, NJ = 96; and for Australian specimens (AUST): MP= 100, NJ = 99.

Fig. 1. One of two most parsimonious trees inferred from MP analysis of the ITS region dataset for cultures from 10 specimens. Numbers at the internal nodes indicate MP and NJ bootstrap values, respectively.

Morphological studies corroborated previous descriptions based on basidiomes from both sides of the Pacific (Rajchenberg, 1994). No significant differences between Argentinean and Australian specimens were observed in terms of pores size, basidiome size and consistency, hyphal construction of the basidiome, hyphal features including branching, variable width and wall thickness, basidia, and spores shape and size; all features were as recorded previously [Rajchenberg (1994, 2006); cfr. also Reid (1957) and Cunningham (1965)]. In particular, basidiospores showed homogeneity in being obovate, broad-ellipsoid to drop-shaped, with thickened walls and similar in size. A statistical analysis of spore sizes did not show grouping of specimens according to geographic distribution (Table 2).

Ecological differences, i.e. differences in substrates between specimens from Argentina and Tasmania were not detected through the phylogenetic analyses.

Discussion

Results of this study show that populations of R. cretacea from Australia and southern Argentina could be considered as distinct at the species level on phylogenetic grounds. The bootstrap values that support both sub-groups are high enough to sustain this conclusion and accord to a phylogenetic species concept. Nevertheless, the typological and biological species concepts do not align with our phylogenetic results. Detailed morphological study failed to identify morphological differences between specimens from the two widely separated geographic regions; on the contrary our results corroborated previous descriptions that included specimens from Australia, southern Argentina andsouthern Chile. In addition, mating tests by Rajchenberg (1994), when he introduced the genus Ryvardenia, suggested one biological species. In that study three tissue/polysporic cultures from Tasmania and Victoria (Australia), that were also used in the present phylogenetic study, were confronted against 6 monosporic cultures from basidiomes MR 10621 (Argentina, Prov. Chubut, Sierra Colorada, M. Rajchenberg 10621¸1-IV- 1992, on standing Nothofagus pumilio, at CIEFAP herbarium) and MR 10674 (Argentina, Prov. Neuquén, Parque Nacional Lanín, Lago Hermoso, margin N, M. Rajchenberg 10674, 11-IV-1992, on fallen branch of Nothofagus dombeyi, at CIEFAP herbarium). These confrontations gave positive results after 7 weeks and, therefore, gave support to the idea that the specimens are conspecific from a biological point of view.

Several examples of non-congruence between the biological and phylogenetic species concepts are present in the mycological literature and it is the taxonomist's decision to recommend one or the other. Among only a few examples from the Southern Hemisphere, Pildain et al. (2009, 2010) showed for Armillaria that A. novae-zelandiae (G. Stev.) Herink forms a clade with specimens from southern Argentina and Chile, New Zealand, Australia and Malaysia, but there was a strong substructure formation with four subclades, each corresponding to geographically different areas. Strains from Patagonia grouped together and apart from those of other geographic regions in a well supported subclade, suggesting the possibility that they represent a distinct taxon. However, based on morphology they were similar to specimens from New Zealand and additional (i.e., biological) evidence is considered necessary before describing new species in that group. Paulus et al. (2000) also found a geographically based, phylogenetic substructure for populations of the polypore Schizopora radula (Pers.: Fr.) Hallenb. from southern Argentina, New Zealand and Australia. In this case biological intercompatibility was also demonstrated, precluding further taxonomic differentiation at the species level.

Geographically delimited populations of a fungal species may often be distinguished phylogenetically (Hallenberg et al. 2007). This may be due to their long history of isolation (Jin et al. 2001) as might be the case for many austral fungal species. A vicariant event such as the separation of Gondwana and the following tectonic drift of land masses may have dominated their history. One should be warned, though, that trans-oceanic dispersal could also have taken place during the millions of years of progressive separation, as was documented by Sanmartín & Ronquist (2004) and Muñóz et al. (2004) for the austral biomes. Maintenance of intercompatibility (i.e., breeding processes) could be explained by such more recent events, but also by the lack of need to develop reproductive isolation due to the geographic isolation. Because species are biological entities of relevance in ecological, pathological and biodiversity contexts, it is better to understand them as species complexes when no morphological, ecological and/or compatibility differences exist. For this reason it is our conclusion that R. cretacea is a species that is undergoing a speciation process as evidenced by the existence of two phylogenetic lineages. This work is a first step to assess the relationships within this taxon. More research including monosporic x monosporic compatibility tests and multigenic phylogenetic studies are necessary to further understand the taxonomic circumscription of this complex.

Acknowledgments

This paper is dedicated to Prof. Leif Ryvarden in honor of his numerous contributions to Polypore taxonomy, his untiring collecting activity throughout the world, and in recognition to his open and kind spirit. Thanks for that Leif! This study was funded by PIP-CONICET 80101000. The authors are members of the "Carrera del Investigador Centífico", National Research Council of Argentina (CONICET). Dr. P.K. Buchanan (Landcare Research, Auckland, New Zealand) kindly arranged the shipment of specimens kept at PDD. He and an anonymous reviewer made most valuable suggestions to improve the manuscript.

References

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